Impaired phagocytosis in agrin-deficient macrophages. (A) Confocal images and phagocytic index of CD11b⁺/F4/80⁺ macrophages sorted from control or Musk-L;Agrn^−/− spleens and incubated with opsonized Alexa Fluor 594–conjugated zymosan A bioparticles (red) for 1 hour at 37°C (green: Alexa 488–Falloidin). The phagocytic index represents the number of red particles per macrophage. Scale bar, 10 μm. (B) Representative images and phagocytic index of an in vivo phagocytosis assay. Irradiated recipients (CD45.1) reconstituted with control or Musk-L;Agrn^−/− BM cells 9 weeks after the transfer were injected intratraperitoneally with 5 × 10⁷ heat inactivated FITC-labeled conidia and killed 30 minutes later. Peritoneal macrophage phagocytosis was analyzed by FACS on the CD45.1⁻/CD11b⁺/F4/80⁺ population. Alternatively, cytospins were prepared and stained with Diff Quick (images). Scale bar, 10 μm. (C) CD11b⁺/F4/80⁺ macrophages sorted from ctrl and Musk-L;Agrn^−/− were plated onto hIgG-coated coverslips and fixed after 60 minutes. Representative images are shown; scale bar, 10 μm. Analyses of adherent membrane surface area (μm²) and phalloidin MFI are shown. (D) F-actin response (phalloidin staining, green) in peritoneal macrophages during formation of phagocytic synapses with opsonized zymosan A bioparticles (red) for 1 minute. Representative images and statistical analysis of the phagocytic cup area (μm²) are shown. Scale bar, 10 μm. (E) Resident peritoneal CD11b⁺/F4/80^high macrophages sorted from control mice were treated with 10 μg/mL of soluble, recombinant agrin (1153-1959; R&D) or (1379-1940; kindly provided by Panos Kabourdis, William Harvey Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London), as indicated. Induction of actin polymerization (phalloidin staining) was analyzed by confocal microscopy (left) and flow cytometry (right). Scale bar, 5 μm. Error bars represent SEM and *P ≤ .05, **P ≤ .01, ***P < .0001.